Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Edited by Dennis R. VoelkerMammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (⌬⌿ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissuespecific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ⌬⌿ m hyperpolarization, a known cause of ROS and trigger of apoptosis.

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H 2 O 2 . Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.-Kalpage, H. A

Cytochrome c is a functionally diverse protein, carrying electrons in the electron transport chain and playing a critical role in cellular apoptosis. Previous work by our lab suggest the importance of reversible phosphorylation of cytochrome c in regulating its functions. We hypothesize that under healthy conditions, phosphorylated cytochrome c partially inhibits mitochondrial respiration and maintains healthy mitochondrial membrane potential, preventing ROS generation, while cellular stress‐mediated dephosphorylation leads to increased respiration and ROS generation, initiating apoptosis.In this study, we purified bovine kidney cytochrome c in the presence of phosphatase inhibitors, identified threonine phosphorylation by immunoblot analysis, and determined threonine 28 phosphorylation by Nano/LC/ESI/MS/MS. To characterize its effect, Thr28 was mutated to glutamate, a phosphomimetic mutation, and alanine, a nonphosphorylatable control, and experiments were performed with bacterially overexpressed proteins. In the reaction with cytochrome c oxidase, Thr28Glu showed significant reduced respiration rates compared to wild‐type, with no alteration in caspase‐3 activation. Thr28Glu had a higher reduction rate from an initial oxidized state, with no change in the rate of oxidation compared to wild‐type, and was found to be resistant to heme destruction in the presence of excess ROS. Our data suggest that Thr28, conserved in mammalian cytochrome c, is an important regulatory site.

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